Brain becomes more resilient to injury through activity, including that from spreading depression (SD). While, mechanisms for this intrinsic process remain undefined, they involve innate cytokines. Importantly, innate cytokines are redundant and pleiotropic so multiple cytokines can have similar effects and a single cytokine can have variable effects. This is due to complex interactive signaling at the level of ligands, receptors, and phosphoproteins. Further complexity stems from the diversity of brain cell types, with each capable of unique expression changes. To date such complexity has precluded their systematic study. We will resolve this void via combination of two strategies to accomplish the general goal of defining the innate cytokine signature of SD-induced neuroprotection. The first uses simultaneous, multiplexed measurement of innate cytokine pathway variables (i.e., cytokines, receptors, phosphoproteins). The second applies classical biochemical pathway dissection strategies to mimic (by application of recombinant cytokine ligands) and modulate (by application of cytokine pathway inhibitors) the biological response to NMDA injury or its alteration by SD. Studies will be performed in hippocampal organotypic cultures (HOTCs), which resemble their in vivo counterparts;lack evidence of confounding inflammatory changes and are isolated, and thus, are not influenced by systemic immune changes. Our data show that HOTCs undergo NMDA-mediated injury, SD, and SD-induced neuroprotection like that seen in vivo. Furthermore, this neuroprotection depends on cytokines, via compensatory, adaptive responses. Coupled with novel tools, HOTCs are well suited to our specific aims of defining innate cytokine: 1) ligand;2) receptor;and 3) related phosphoprotein changes associated with SD-induced neuroprotection from NMDA injury compared to NMDA injury alone - as a means to establish an encompassing signature of the cellular and tissue innate cytokine pathway changes that evoke SD-induced neuroprotection. These aims will be accomplished using: newly developed microsphere-based flow cytometric assays to simultaneously measure innate cytokines (IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-10), their receptors (IL-1R1, TNFR1, IL-6Ralpha/gp130, IFNgammaR, IL-10R), and related downstream phosphoprotein (kinases (ERK1/2, P38MAPK, JNK) and transcription factors (ATF-2, NFkappaB, STAT3)). Changes will be localized to cells using multiplexed real-time RT-PCR of mRNA derived from laser dissection microscopy acquired specific cell enhanced samples (cytokines) and double label immuno- histochemistry (receptors and phosphoproteins). Innate cytokines trigger altered function via gene activation. We suggest that identification of compensatory changes of the innate cytokine system will predict which genes, and related protein pathways, within the system (and within other signaling systems) will be optimal therapeutic targets to emulate protective capacities of intrinsic neuroprotection.